Progress 2016-2017 I Identification of Novel factors contributing to repair of Topoisomerase 2 DNA-protein crosslinks Topoisomerase 2 (TOP2) DNA transactions are essential for life, and involve the formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to targeting by potent anticancer TOP2 drugs. Repair of TOP2 DNA-protein crosslinks can be initiated by proteosomal degradation, but if alternative non-proteolytic direct resolution pathways for these highly genotoxic lesions occurs is unknown. With proteomics screening approaches, we identified ZATT (Zinc finger protein Associated with TDP2 and TOP2), a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT directly binds TOP2 and licenses a proteasome-independent Tyrosyl DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. Further, a ZATT SUMO2 E3/E4 ligase activity regulates TDP2 interactions with sumoylated TOP2, driving efficient mobilization of TDP2 to DNA damage through a novel TDP2-SUMO2 split-SIM engagement platform. Altogether, these results delineate a ZATTTDP2 catalyzed and SUMO2-modulated pathway for the direct resolution of TOP2cc, with implications for cancer therapy and chemoresistance. II Structure and function of the Ape2 nuclease The Xenopus laevis APE2 nuclease dictates 3-5 nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. We have determined that APE2 resection activity is modulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DNA damage response proteins Topoisomerase 3 and NEIL3 DNA glycosylase, as well as transcription and RNA regulatory proteins such as TTF2, TFIIS and RPB9. Biochemical and NMR results establish the nucleic acid binding activity of the Zf-GRF domain. Further, APE2 X-ray structures and small angle X-ray scattering (SAXS) analyses show that the Zf-GRF fold is typified by a crescent shaped ssDNA binding claw, that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3-5 exonuclease processing, as well as prevent efficient APE2-dependant RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module dictating the key ssDNA strand break resection function of APE2, and further reveal topological similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9. III Surveying the structure and function of CtIP/Ctp1/Sae2 Vertebrate CtIP, and its fission yeast (Ctp1), budding yeast (Sae2) and plant (Com1) orthologs have emerged as key regulatory molecules in cellular responses to DNA double strand breaks (DSBs). By modulating the nucleolytic 5-3 resection activity of the Mre11/Rad50/Nbs1 (MRN) DSB repair processing and signaling complex, CtIP/Ctp1/Sae2/Com1 is integral to the channeling of DNA double strand breaks through DSB repair by homologous recombination (HR). Nearly two decades since its discovery, emerging new data are defining the molecular underpinnings for CtIP DSB repair regulatory activities. CtIP homologs are largely intrinsically unstructured proteins comprised of expanded regions of low complexity sequence, rather than defined folded domains typical of DNA damage metabolizing enzymes and nucleases. A compact structurally conserved N-terminus forms a functionally critical tetrameric helical dimer of dimers (THDD) region that bridges CtIP oligomers, and is flexibly appended to a conserved C-terminal Sae2-homology DNA binding and DSB repair pathway choice regulatory hub which influences nucleolytic activities of the MRN core nuclease complex. The emerging evidence from structural, biophysical, and biological studies converges on CtIP having functional roles in DSB repair that include: 1) dynamic DNA strand coordination through direct DNA binding and DNA bridging activities, 2) MRN nuclease complex cofactor functions that direct MRN endonucleolytic cleavage of protein-blocked DSB ends and 3) acting as a protein binding hub targeted by the cell cycle regulatory apparatus, which influences CtIP expression and activity via layers of post-translational modifications, protein-protein interactions and DNA binding.